Multichannel, multipurpose sample collection and drug delivery system for laboratory animals

ABSTRACT

A multichannel, multipurpose sample collection and drug delivery system for laboratory animals includes several sampling and/or infusion devices mounted on a powered turntable above the cage but out of the animal&#39;s sight. Probes are constructed using a unique four-channel design: this permits (a) the periodic infusion of drugs without the possibility of artifacts due to pressure shocks and (b) the addition of preservatives to the sample at the site of its collection. A simplified sample collection device stores samples in a coil of PTFE tubing, and a constant-pressure perfusion system serves microdialysis probes. Further advantageous embodiments of the present invention include (a) systems to measure feeding, drinking and motor activity; (b) automated and unattended systems for blood withdrawal and drug delivery; (c) a data link with a personal computer to provide for data acquisition and more sophisticated control; (d) an improved method for sampling extracellular concentrations of neuropeptides, which are difficult to measure with conventional microdialysis; (e) the capability to perform electrophysiology simultaneously with microdialysis (or other sampling methods); and (f) the adaptation of the system for use with animals larger or smaller than the rat.

BACKGROUND OF THE INVENTION

The present invention relates generally to sample collection and drugdelivery systems, and more particularly to sample collection and drugdelivery systems for freely-moving laboratory animals.

Most animal studies in physiology, and many in pharmacology and basicbiology, require samples (e.g. blood or cerebrospinal fluid (CSF)) to beremoved for analysis, some sort of electrical activity to be monitored,and/or drugs to be administered at precise times. If such experimentsare to be conducted in alert, freely-moving animals, which is generallypreferred, some sort of tether encasing the fluid or electricalconnections to the animal is normally required. To allow improvedfreedom of movement, a swivel is generally provided between the animaland the sampling or infusion device. Multichannel electrical swivels maybe reasonably satisfactory (although mercury-based devices arehazardous), but fluid swivels are often problematical, being stiff andsubject to leaks and blockages. This restricts the animal's movement andrequires frequent experimenter intervention to remove twists from thetether. The result is that the ideal of a stress-free environment forthe animal under study is difficult or impossible to achieve withcurrently available equipment.

There is no commercially available equipment that provides thecapability of multichannel fluid sampling and/or administration inalert, tethered animals. However, an alternative method for achievingmultichannel sampling has been published (See Matsumura, H., Kinoshita,G., Satoh, S., Osaka, T. and Hayaishi, O., "A novel apparatus thatpermits multiple routes for infusions and body fluid collections in afreely-moving animal," J. Neurosci. Methods 57 (1995) 145149). In thatsystem, the rat itself, rather than the supporting equipment, is movedon a turntable to reduce the twisting of the tether. This approach isundesirably stressful to the animal and presents other problems.

The present invention is therefore directed to the problem of developinga sample collection and drug delivery system with a unique multichannelcapability for use with alert, freely moving laboratory animals, withoutputting any additional stress on the laboratory animal.

SUMMARY OF THE INVENTION

The present invention solves this problem by mounting the samplingand/or infusion devices on a powered turntable above the cage but out ofthe animal's sight. Rotation of the tether as the animal moves isdetected by magnetic switches which cause the turntable to turn in thedirection taken by the animal. By this means, the need for troublesomefluid or electrical swivels is entirely eliminated, and the animalexperiences almost zero torque on the tether.

Because it is the swivel that otherwise limits the number of fluid orelectrical connections in traditional systems, eliminating the swivelremoves this constraint on the number of connections. As a result, it isnow possible to place multiple probes in alert, freely moving animals inorder to sample several brain regions at the same time, such as to makesimultaneous measurements of monoamine release in the ventral tegmentalarea and in its dopaminergic projection regions. Previously, multiplesimultaneous measurements at different sites could only be achieved byanesthetizing or constraining the animal, which was not satisfactory.

Moreover, probes can now be constructed using a unique four-channeldesign: this permits (a) the periodic infusion of drugs without thepossibility of artifacts due to pressure shocks and (b) the addition ofpreservatives to the sample at the site of its collection.

The present invention also includes a simplified sample collectiondevice in which samples are stored in a coil of PTFE tubing, and aconstant-pressure perfusion system for microdialysis probes.

Further advantageous embodiments of the present invention include (a)systems to measure feeding, drinking and motor activity; (b) automatedand unattended systems for blood withdrawal and drug delivery; (c) adata link with a personal computer to provide for data acquisition andmore sophisticated control; (d) an improved method for samplingextracellular concentrations of neuropeptides, which are difficult tomeasure with conventional microdialysis; (e) the capability to performelectrophysiology simultaneously with microdialysis (or other samplingmethods); and (f) the adaptation of the system for use with animalslarger or smaller than a rat.

One advantageous embodiment of the apparatus for couplinginstrumentation to the animal employs a rotatable platform with theinstrumentation mounted on the platform. Thus, the tether is connectedboth to the laboratory animal and the instrumentation. In addition, arotation compensator detects rotation of the tether and controlsrotation of the platform based on the detected rotation.

In this embodiment, the rotation compensator uses a magnet mounted onthe tether, a power source connected to the rotating platform, and apair of magnetic switches mounted in a common plane with the magnet sothat rotation of the magnet away from one magnetic switch is towards theother magnetic switch. A second power source is connected to themagnetic switches, with an impedence network in parallel with therotating platform. A pair of relays are used to switch the polarity ofthe current from the power source to the motor based on the detectedrotation.

Another embodiment of the present invention employs a coupling betweenthe tether and the instrument. The coupling has a tube through which thetether passes, a magnet mounted on the tube, and a rotation stop juttingout from the tube, which limits the amount of the tube's rotation.

Yet another advantageous implementation of the turntable apparatus ofthe present invention includes a force minimizing mechanism that adjuststhe length of the tether in the cage to account for slack created byanimal movement. When the animal moves closer to the tether the lengthadjustment pulls on the tether to reduce the excess length of the tetherwithin the cage. When the animal moves away from the tether i.e., pullson the tether, the length adjustment mechanism lets the length of thetether increase to provide freedom of movement for the animal.

One implementation of the force minimizing mechanism uses a balance armto which one end of the tether is mounted, and a counterweight connectedto the balance arm via a pair of pulleys and a cable. As the animalmoves, the force on the tether either increases or decreases, whichcauses the counterweight to move up and down in reaction to the changein force, respectively, which lowers or raises the balance arm, causingthe length of the tether in the cage to lengthen or shorten,respectively.

To fine tune the acceleration and deceleration of the turntable, oneadvantageous embodiment of the turntable employs an array of magneticsensors providing positional information about the tether, a steppermotor drive controlling the rotatable platform, and a microcomputercontrolling the stepper motor drive based on the positional information.

In this case, the apparatus includes a means for altering the stepfrequency of the stepper motor, so that smoother acceleration anddeceleration is achieved and a maximum rotational speed can be madeproportional to the displacement. One possible implementation of thismeans is a microcomputer to control the step frequency of the steppermotor drive.

Another alternate embodiment of the turntable apparatus includes a cagefor containing the animal, and the rotatable support mechanism ismounted above the cage. In this embodiment, a computer compiles reportsregarding movement by the animal and infrared transmitters shineinfrared beams through the cage which are detected by infrared sensorsmounted around the cage. These sensors are coupled to the computer andindicate when the beams are interrupted. As a result, these beamscontain information about the animal regarding horizontal movement,rearing, and access to food and water, which information is passed tothe computer. The computer then outputs reports correlating activitydata with rotational activity data detected by rotation of the rotatablesupport mechanism.

According to the present invention a method for coupling a tether to alaboratory animal includes detecting a rotation of the tether, adjustingan orientation of the tether relative to the animal based on thedetected rotation, and modifying the length of the tether (or itsprojection into the cage) to account for movement of the laboratoryanimal. Additional advantageous steps include mounting a slackadjustment device on a rotatable platform, and rotating the platformaccording to the detected rotation simultaneously with changing theslack in the tether.

Another aspect of the present invention is a sample collection device.This novel device includes a coil with one end connected to the samplingsite, and a pump controlled by a timer, which at preset intervals drawsa sample into the coil followed by a bubble of gas to keep successivesamples separate. Since the device operates automatically, it isdesirable to include a cooling device surrounding the coil to preventthe samples from becoming unusable. One particularly advantageouscooling device is Peltier cooling device, or a reverse thermocouplecooling system. A computer controls the cooling device based oninformation provided by a thermistor sensor.

For those samples that might be tainted by air, the present inventionemploys a gas reservoir at one end of the coil. The gas source can useany of the noble gases, or a mix of them.

To control the size of the bubbles, even after many samples, the presentinvention uses a solid-state delay relay maintaining successive samplesseparate by introducing air bubbles at regular intervals. The relay iscontrolled by a computer, which controls the frequency of bubbleintroduction. Sensors detecting the size of the bubble are used toaccurately introduce bubbles into the coil.

An automated fluid sampling system according to the present inventionfor obtaining samples from a living organism employs the coil, and anindwelling cannula attached to the living organism. The indwellingcannula has a main cannula with a tip, a body and an concentric innercannula. One syringe is attached to the one end of the coil, anothersyringe is filled with an anticoagulant and is attached to theindwelling cannula via the concentric inner cannula. The second syringedelivers the anticoagulant via the concentric inner cannula to a pointabout 1 mm from the tip of the main cannula. Yet another syringe isattached to the indwelling cannula via the body of the indwellingcannula, and delivers a preservative solution into the body of thecannula. An external computer transmits an activation signal to threestepper motors, which operate the three syringes. A microcomputerreceives the activation signal from the external computer and controlsthe three stepper motors according to the activation signal.

In the above system, it is particularly advantageous if the system usesthe following withdrawal sequence. First, the sample syringe withdraws asample at a rate faster than the delivery rate of the anticoagulant andpreservative, whereby a total volume withdrawn=volume ofpreservative+volume of anticoagulant+internal volume of cannula+samplevolume. To backfill the cannula, the sample syringe is withdrawn at awithdrawal rate=coagulant delivery rate, wherein total volume=internalvolume of cannula. To perform an anticoagulant drip, there is no mainsyringe or preservative syringe motion, and the anticoagulant flowrate=about 1 μliter per minute by intermittent pulsing of theappropriate stepper motor. In this case, samples are stored in thesample coil between bands of anticoagulant.

An automated drug delivery system for delivering a drug to a livingorganism according to the present invention employs an indwellingcannula attached to the living organism and one end of the coil, with atee coupling at one end. Two computer controller stepper motors activatethe two syringes, one of which is maintained at a cold temperature andcontains a solution of the drug and the other syringe contains normalsaline. Both syringes are connected to the cannula by the tee. In thisinstance, delivery sequence is: as follows. First, a required volume ofdrug solution is delivered into the cannula, followed by delivery ofsaline into the cannula equivalent to 110% of the internal volume of thecannula. In this embodiment, the cannula has a diameter less thanapproximately 40 μm, and the computer controls the flow rates to be inthe range of approximately 1 μliter per minute.

Another aspect of the present invention includes an apparatus forcollecting a fluid sample from a living organism and for providing drugsto the living organism. This apparatus includes a membrane attached tothe living organism, and a multichannel microdialysis probe connected tothe membrane. The first channel delivers perfusate to the sampling siteon the living organism and extends into the membrane. The second channeldelivers a preservative to mix with the fluid sample immediately afterthe fluid sample enters the probe. A third channel receives the fluidsample from the living organism, and has a relatively wide bore ascompared to the other channels. A fourth channel provides a means ofinjecting a drag into the perfusate inflow in order to deliver the dragto the animal via the microdialysis membrane.

By connecting a coil of flexible tubing to the third channel of themicrodialysis probe, and the time controlled pump of the presentinvention to one end of the coil, one obtains a unique sampling systemaccording to the present invention. In this system, at preset intervalscontrolled by the timer, the pump draws a sample into the coil followedby a bubble of gas to keep successive samples separate.

Another aspect of the present invention is an apparatus for supplyingliquid to a probe. This apparatus includes a pressurized container witha pressure sealed exit port, at least one reservoir of liquid, at leastone tube extending into the liquid in the reservoir and exiting thecontainer via the sealed port, which is attached to the probe at itsother end. In this case, the tube has a very narrow inner diameter sothat the volume of the container is several orders of magnitude greaterthan the volume of the preservative. The tube also has a predeterminedlength greater than approximately 40 millimeters and the container has apressure less than approximately 15 pounds per square inch.

A multichannel perfusion device according to the present invention forsupplying preservative to a plurality of microdialysis probes hasseveral tubes attached to the probes, each tube has a predetermined andfixed length, and a relatively small diameter. Several reservoirs ofpreservative are located in the container. Each of the tubes extendsinto the preservative in one of the reservoirs. The container ispressurized at a low pressure has a pressure sealed exit port throughwhich the tubes exit the container, whereby the pressure of thecontainer relative to the length and diameter of the tubes limits thepressure of the fluid in the tubes. In this device, it is particularlyadvantageous if the pressure of the container is between approximately 5and 15 psi, the length of the tubes is between approximately 40 and 300mm, the diameter of the tubes is approximately 40 μm, the flow rate fromthe tubes is less than approximately 4 μliters per minute, and thevolume of the container relative to the volume of the preservative beingdischarged is such that a maximum pressure that can exist in the tubesis less than approximately 10 pounds per square inch.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a diagram of a turntable mounted on a cage with atethered rat according to the system of the present invention.

FIG. 2 is a schematic of the turntable and cage apparatus of the presentinvention.

FIG. 3 depicts a close-up of the five-channel electrical swivel anddrive mechanism used in the apparatus of the present invention.

FIG. 4 shows one embodiment of the apparatus of the present invention,specifically including pumps and sample collectors of the presentinvention mounted on the turntable of the present invention.

FIG. 5 depicts a detail of the sensor head used in the apparatus of thepresent invention.

FIG. 6 depicts a sample collection coil of the present invention.

FIG. 7 depicts a schematic of a four-channel microdialysis probe of thepresent invention.

FIG. 8 is a graph of the rate of flow of artificial CSF throughincreasing lengths of microbore fused silica tubing.

FIG. 9 depicts a constant-pressure perfusion system according to thepresent invention.

FIG. 10 shows one embodiment of the sample loop of the presentinvention.

FIG. 11 shows a top view of the counterbalanced arm and sensor head.

FIG. 12 depicts a side view of the rotation detector.

FIG. 13 depicts a section of the side view of FIG. 12.

FIG. 14 depicts the electrical schematic of the microdialysis turntableaccording to the present invention

FIG. 15 depicts another close-up view of the turntable system shown inFIG. 2.

DETAILED DESCRIPTION

The multichannel automatic sample collection and drug delivery system ofthe present invention has several potential applications. The generalaim has been to produce a highly reliable automated multichannel samplecollection system at a cost well within that of current commercialequipment.

One part of the present invention, i.e., the turntable, provides anelegant solution to many of the problems associated with physiologicalor pharmacological studies of freely-moving laboratory animals andprovides a significant advantage in any experiment in which animals mustbe tethered in order to obtain samples of body fluids, administer drugson a precise schedule or measure electro-physiological responses.Moreover, because the apparatus of the present invention permits freemovement of the animal under study, it has a much broader range ofapplications than the conventional equipment that it is designed toreplace. For example in microdialysis, which is performed in anestimated 500 to 1000 laboratories, many studies are still performed inanesthetized animals when alert, freely moving animals are almost alwayspreferred. The microdialysis probes and tethers of the present inventioncan be used in social situations where commercially available equipment(which is not designed to be nibble-proof) is unsuitable. In fact,almost all microdialysis studies on rats have been performed onsingly-housed animals even though this rodent is a social animal. Theisolation of social animals may act as a stressor, and this may haveinfluenced the outcome and interpretation of some studies.

Another important aspect of physiology that has been largely ignoredbecause of its inconvenience has been the study of circadian variationsin the response to stress or drug administration. The fully automaticturntable makes such studies very easy to perform without disrupting theexperimenter's own circadian rhythms.

Moreover, the application of the turntable device is not limited tothose fields traditionally reserved for microdialysis. For example, inpharmacodynamics, the turntable could be used to assess drug metabolismand tissue penetration in multiple sites simultaneously in chronic aswell as acute studies. In endocrinology, local differences in hormonemetabolism can have drastic effects on their physiological actions.Chronic, simultaneous sampling from multiple tissue sites and brainregions could illuminate many problems concerning the control ofcircadian and seasonal rhythms feedback effects and "end organinsensitivity."

Turntable

A diagram of three turntable apparatuses 1 mounted on a triple cage withthree tethered rats is shown in FIG. 1, and a schematic of a singleturntable apparatus is given in FIG. 2. A close-up view of the schematicshown in FIG. 2 from the base up, without the tether, is shown in FIG.15.

Each turntable apparatus consists of a base 26 supporting an outer frame7, 8 that holds the drive motor 21 and electrical swivel 24, and theturntable itself 4 which is mounted on a raised platform 11 above thebase 26 in order to distance it from the rat 28. The tether 2 comingfrom the rat 28 runs upward through a hole 5 in the center of theturntable 4 and attaches to a counterbalanced arm 12 at point 113.Rotation of the tether 2 produced by movement of the rat 28 is detectedby magnetic switches 133, 134 (see FIGS. 12 and 13) that activate thedrive motor 21 to turn the turntable 4 in the direction of the movementof the rat 28.

A close-up of the five-channel swivel 24 and drive mechanism 33 is shownin FIG. 3. The motor 21 consists of a fractional horsepower reversiblesynchronous motor with a 3:1 step-down belt drive 33 that provides a 20rpm drive to the turntable 4. The electrical swivel 24 employs carbonbrushes and also serves as the upper bearing of the turntable 4. Thefive electrical channels consist of AC live, neutral and ground to theturntable and the CW and CCW AC connection to the synchronous motor 21from relays 136, 137 (see FIG. 14) mounted on the turntable 4. Furtherelectrical and fluid swivel connections could be provided by using ahollow drive shaft in the swivel 24. Alternative means of communicatingwith the turntable data connections might be to use the AC as a carrieror to use infrared signaling.

The rotating surface of the turntable 4 is illustrated in FIG. 15. Atits center is a one-inch hole 5 fitted with a brass tube. The tether 2runs through this brass tube 5 from the cage 27 below. The tube 5revolves in a Rulon bearing mounted in the base platform 11. Thecounterbalanced arm 12 that connects to the tether 2 via the sensor head3 (see FIG. 5) is attached to a counter-weight 25 by means of twooverhead pulleys 23. The weight 25 is constrained within the plastictube 10. The AC-powered syringe pump 42 shown in FIG. 4 is acommercially available model mounted in a vertical position to savespace. An improved and simplified perfusion system 90 for microdialysisis included (see FIG. 9) in an alternate embodiment. The control box 43contains solid-state relays 136, 137 (refer to FIG. 14) that activatethe synchronous motor 21, a DC power supply 135 for the magneticswitches 133, 134 and a commercial timer module 149 to control thesample-collection system by activating the peristaltic pump 45 at presetintervals.

FIG. 5 shows the sensor head 3 that detects rotational movement of thetether 2. The head 3 is mounted on the end 51 of the counterbalanced arm12 by a pair of pivots. The tether 2 is attached to the rotor 53 whichconsists of a brass tube through which infusion lines and samplecollection lines from the rat 28 are routed. A magnet 121 (see FIG. 13)is mounted in a plastic holder 52 on the brass tube 53. The brass tube53 is fitted with upper and lower ball bearings, and a stop 54constrains its rotation to about 270°. At the maximum counter-clockwiseextent of its movement, the magnet 121 (see FIG. 13) on the rotor 53 isaligned with one of the two magnetic switches 133 (FIG. 13). At themaximum clockwise direction, the magnet 121 is aligned with the secondmagnetic switch 134. The sensitivity of the system (i.e. the amount ofrotational movement of the rotor 53 needed to trigger the magneticswitches 133, 134) can be adjusted by moving the magnet holder 52 up ordown the rotor shaft 53.

The turntable system 1 of the present invention is extremely reliable.Some further advantageous embodiments of the present invention includethe following. For example, for simplicity of design, one embodiment ofthe turntable is powered by an AC synchronous motor controlled byrelays. Even using an elastomeric drive belt, this can produce a ratherabrupt acceleration and deceleration of the turntable. One alternativeembodiment includes two changes: (a) an array of magnetic sensors toprovide more precise positional information and (b) a stepper motordrive controlled by a PIC computer chip. By altering the step frequency,smoother acceleration and deceleration is achieved and the maximumrotational speed can be made proportional to the displacement.

Sample Collection Device

The multichannel collection system of the present invention employs aunique device 61 for collecting samples, which is shown in FIG. 6.Another view of the sample coil is shown in FIG. 10. According toanother aspect of the present invention, samples 63 are collected in aloop 64 of 16 Ga Teflon tubing (see FIG. 6) that is housed in aninsulated box 46 (see FIG. 4). The peristaltic pump 45 draws an airbubble 62 into the sample loop 64 to keep samples 63 separate.

The new coil-based sample collector 61 of the present invention is muchsimpler than traditional designs in which the samples are stored inindividual vials. It lends itself to miniaturization and could be usedto advantage in a broad range of applications in which continuoussampling of small volumes of fluid is needed, including chromatographyand electrophoresis. Advantages include low costs of fabrication andoperation as well as protection of samples from evaporation losses andoxidation.

Samples are collected in the open end of a coil 64 of the tubing (seeFIG. 6). At preset intervals controlled by a timer, the peristaltic pump45 (FIG. 4) draws a sample 63 into the sample coil together with abubble of air 62 to keep samples 63 separated.

In the current system, the sample coil is mounted in an insulated box 46(FIG. 4) and kept cool with ice. This works well for about 24 hours. Tomake changing the sample coil 64 easier, an advantageous implementationof this invention employs a Peltier cooling system (i.e., a reversethermocouple). In this case, temperature control is provided by athermistor sensor attached to a PIC computer.

Another embodiment incorporates a helium reservoir at the open end ofthe coil so that oxygen-sensitive samples are separated by a bubble ofinert gas instead of air. Any of the noble gases can be substituted forhelium, if necessary. For certain applications nitrogen may be used.

According to the present invention of the sample collector, samples areseparated by air bubbles introduced at regular intervals determined bythe setting of a solid-state delay relay 149 (FIG. 14). Otheradvantageous embodiments of this invention include the followingmodifications. First, the frequency of bubble introduction can becontrolled more accurately by a PIC computer connected to a number pad.Second, the size of the bubble can be controlled more closely throughthe use of sensors attached to the PIC computer rather than by simplycontrolling the on-time of the peristaltic pump (the latter isunreliable because, as the number of air bubbles in the samplecollection coil increases, so does the resistance to flow--thus bubblesget smaller with time). Another advantageous embodiment substitutes astepper motor-driven syringe for the peristaltic pump, which alsoimproves control.

Microdialysis Probes

Commercially available microdialysis probes are generally designed towork with a single or dual channel swivel and consist of a single inflowand a single outflow line attached to a flexible microdialysis membrane.Because linear flow rates need to be high to reduce degradation ofanalytes in the probe, outflow tubing is usually of very small diameter.This makes the probes prone to blockage (e.g. from particles breakingoff from the seals in the swivel or from the syringe piston) and resultsin a system in which the microdialysis membrane is under positiveinternal pressure. In extreme cases, this causes ultrafiltration of theperfusate and subsequent local tissue damage around the site ofimplantation.

To eliminate these problems, the microdialysis probes 70 of the presentinvention are fabricated so that (see FIG. 7) the outflow tube 72 has arelatively wide bore (250 μm) to reduce the pressure on the membrane 75.The membrane 75 is perfused via tube 74 with artificial CSF, as inconventional systems, but an additional inflow tube 73 deliverspreservative to mix with the sample immediately above the membrane 75.For monoamines, the preservative consists simply of the low-pH mobilephase used for HPLC, but for peptides, peptidase inhibitors, antibodiesor proteins could be added to keep the intact peptide in solution.

A powerful technique in microdialysis is to add a drug or chemical tothe perfusate in order to study its local effects on monoamine release.Normally this is achieved by switching syringes, a procedure that canproduce artifacts through pressure shocks on the membrane. Accordingly,a third inflow line 71 is added to the probe of the present inventionspecifically for the purpose of introducing drugs to the perfusate.

The probe 70 of the present invention is constructed from two sizes ofpolyimide-coated fused silica tubing (PolyMicro Technologies) and fromregenerated cellulose hollow fiber microdialysis tubing (Spectra/Por RC,200 μm i.d., MWCO: 13,000). Delivery tubing is made from PE-10 and fromTSP040105 tubing (40 μm i.d., 105 μm o.d.) in single lengths to avoidjoins. These are glued and sealed into a 40-mm length of TSP250350 (250μm i.d., 350 μm o.d.) as shown in FIG. 7. Active regions are 2.5 mm fordorsal hippocampus and 4 mm for frontal cortex, as described byAbercrombie, E. D. and Finlay, J. M., "Monitoring extracellularnorepinephrine in brain using in vivo microdialysis and HPLC-EC. In T.E. Robinson and J. B. Justice (Eds.) Microdialysis in the Neurosciences,Elsevier, Amsterdam, 1991, pp. 253-274. After drying, these probes 70are then washed through with ethanol and water overnight before beingchecked for leaks, flow rate and in vitro recovery of NE. Ifsatisfactory, the probes 70 are mounted into a tether assembly whichconsists of Peek tubing attached to a swivel at one end and fitted witha "Soft-Touch" HPLC nut at the end that attaches to the guide cannula.

Much of the work currently being conducted or planned in laboratoriesinvolves subtle physiological responses to relatively mild stresses suchas noise or the presence of conspecifics. Normal responses to low levelstresses can only be detected in the absence of chronic stresses (seeWeiss, J. M., Glazer, H. I., Pohorecky, L. A., Brick, J. and Miller, N.E. "Effects of chronic exposure to stressors on avoidance-escapebehavior and on brain norepinephrine," Psychosom. Med. 37 (1975)522-533). Therefore, a basic requirement of the turntable system wasthat it should provide a nonstressful environment for the rat. Thecriteria for its meeting this requirement are as follows: 1) weightchanges in animals in the turntable apparatus must be no different fromthose housed in the colony; 2) rats in the turntable apparatus mustexhibit normal circadian patterns of feeding, drinking and motoractivity (normally most of this activity is displayed during specificperiods of the dark phase in these nocturnal animals); 3) rats in theturntable apparatus must show normal circadian rhythms of corticosteronesecretion (high levels early in the dark phase) and also exhibitincreased corticosterone concentrations and increased brainextracellular concentrations of monoamines and monoamine metabolite inresponse to mild stresses such as noise or the presence of humans. Theturntable apparatus of the present invention meets these criteria.

Constant Pressure Perfusion Device

The constant-pressure perfusion device 90 of the present invention shownin FIG. 9 provides a low cost alternative to syringe-pumps and possessesmany advantages over the latter under circumstances in which flowresistance is low and relatively constant.

Commercially available syringe pumps used on the turntable for perfusingthe microdialysis probes work satisfactorily for periods of up to 24hours, but at that time, the syringes have to be reloaded. Thealternative of using larger syringes and slower pump motors provedunsatisfactory because of poor flow control. Unfortunately, the processof changing a syringe can lead to artifacts created when abrupt pressurechanges cause swelling or contraction of the microdialysis membrane,thereby displacing brain tissue, and can also introduce particles intothe system that are capable of blocking the microbore tubing used in theprobes.

To overcome this problem, the inventors adopted an approach based onfluidic theory. The familiar Ohms Law of electronics (current=voltagedivided by resistance) has its equivalent in fluidics: flow=pressuredivided by resistance. To obtain the very slow flow rates used inmicrodialysis, all that is needed is a source of constant pressure and aresistor in the form of a fixed length of very narrow-bore fused silicatubing (FST: 40 μm i.d.). The relationship between flow rate and tubinglength is illustrated in FIG. 8. The constant pressure perfusion device90 of the present invention (FIG. 9) includes a helium-chargedpolypropylene bottle 94 containing reservoirs 95 of artificial CSF andpreservative sufficient to perfuse one or more probes for several days.The lengths of FST dip directly into the fluid and exit through ports 93in the side of the bottle 94 where they are attached to the respectiveinflow lines of the probes. Because the volume of the bottle 94 isseveral orders of magnitude greater than the volume of the liquiddischarged, the pressure in the bottle 94 remains steady.

An important consideration in the design of perfusion systems formicrodialysis is the safety of the animal. Conventional syringe pumpsare capable of generating pressures of the order of several atmospheres.If the outflow from a microdialysis probe becomes blocked (a notinfrequent occurrence with conventional designs), this entire pressureis then applied to the fragile dialysis membrane, which may burst withinthe animal's brain. With the constant pressure pump 90 of the presentinvention, the maximum pressure that can be applied to the membrane isonly 5 or 10 psi, which is not sufficient to burst a membrane. Not onlyis this multichannel perfusion device 90 far simpler and less costlythan commercially available syringe pumps but, with respect to the needsof microdialysis, performance is in many respects superior.

Infrared Activity Monitors

In order to be able to relate neurochemical events to behavior,according to another aspect of the present invention, the cages areequipped with infrared beams to monitor horizontal movement, rearing,and access to food and water. The infrared system is controlled by a PICcomputer with total beam breaks displayed on LCD panels. For moresophisticated analysis, however, the raw activity data is uploaded to apersonal computer and is combined with the rotational activity datadetected through the tether.

Automated Blood Withdrawal System

Although microdialysis offers many advantages over blood withdrawal formeasuring hormone or drug levels, some hormones, notably peptidehormones, cannot be measured using current methods of dialysis. Manualblood sampling is very stressful to the animal and cannot be used forfast-reacting substances such as blood catecholamines and ACTH. Remotesampling systems using indwelling venous or arterial cannulae have beenmuch used for measuring stress hormones and circadian endocrine rhythmsthat would be disrupted by manual methods.

According to the present invention, a fully automated blood samplingsystem can be used in conjunction with the turntable to solve thisproblem. The main problem in remote sampling is blockage of the cannulaeby blood clots--particularly small fibrous masses that form at the endof the cannula and act as a one-way valve. The normal method ofpreventing clot formation is to backfill the cannula with anticoagulantbetween samples and to provide a very slow anticoagulant drip to keepthe cannula tip free without heparinizing the whole animal. The presentinvention uses three stepper motor-driven syringes and a cooled samplecoil to store the samples (see above). The indwelling cannula isattached to one end of the coil and the main withdrawal syringe isattached to the other end. A second syringe is filled with anticoagulantwhich it delivers via a concentric inner cannula to a point about 1 mmfrom the tip of the main cannula. The third syringe delivers a suitablepreservative solution (e.g. acid, antioxidant, peptidase inhibitor oreven antibody) into the body of the cannula. All stepper motors arecontrolled by a PIC computer activated by an external PC via infraredsignals. The withdrawal sequence proceeds as follows:

(1) sample withdrawal--the main syringe withdraws at a rate faster thanthe delivery rate of the anticoagulant and preservative. In this case,total volume withdrawn=volume of preservative+volume ofanticoagulant+internal volume of cannula+sample volume.

(2) cannula backfill--the main syringe withdrawal rate equals coagulantdelivery rate. In this case, total volume=internal volume of cannula;

(3) anticoagulant drip--no main syringe or preservative syringe motion.In this case, the anticoagulant flow rate=about 1 μl/min by intermittentpulsing of the stepper motor.

Blood samples prepared in this way are stored in the sample coil betweenbands of anticoagulant. Because the blood will be diluted withanticoagulant and preservative, extraction may be necessary beforeanalyzing the component of interest. Results can be expressed relativeto the hemoglobin or creatinine concentration.

Automated Drug Delivery

The prototype turntable and tether system described above alreadyincorporates a cannula which is used for the remote intraperitonealdelivery of drugs. However, this has to be performed manually by theexperimenter, which necessitates briefly inactivating the turntable andinjecting the drug into the cannula, which is sometimes at inconvenienttimes of night. An automated system is preferred.

The present invention provides a device consisting of two stepper-motoractivated syringes controlled by a PIC computer. One syringe (whichcould be kept ice cold if necessary by fluid from the sample collectorcooling system) contains a solution of the drug; the second syringecontains normal saline (or artificial CSF for intracerebral delivery)and both are connected to the head of the cannula by a Tee. In thiscase, the delivery sequence consists of the following:

(1) delivery of the required volume of drug solution into the cannula;

(2) delivery of a volume of saline into the cannula equivalent to 110%of the internal volume of the cannula.

By this means diffusion of drug out of the cannula between deliveryepisodes is avoided. To keep volumes low enough for intracerebraldelivery, the cannula is constructed from FST (40 mm inner diameter) andflow rates are in the range of 1 μl/min.

Data Communication with a Dedicated PC

The PIC computers that are used to control the turntable movement,sample collector and activity monitor are extremely cheap (less than$50) and are designed to be very rugged and reliable. The turntable isfully functional without the need to attach it to a PC, however, formore sophisticated timing of drug infusions and sample collection, thepresent invention also includes a PC interface. In this arrangement,several PIC computers are linked together via a linear token networkthat can communicate with a main PC via its serial communications port.

The PIC computer on the turntable requires a more specialized interfaceconsisting of a two-way infrared transmission link. Customized softwareon the PC (possibly running within a spreadsheet) gathers activity data,information on the amount of rotational movement, and provides acountdown to the next sample "change." Timed infusions of drugs via thedialysis probes or via indwelling catheters can also be controlled bythe PC.

Methods for Sampling Neuropeptides

Measuring release of neuropeptides in the brain has importance to manyfields of neuroscience, including neuroendocrinology, neuroimmunologyand the study of feeding behavior and reward systems. Unfortunately,microdialysis has not provided an easy way to do this because rates ofrecovery of most peptides are very low. The inventors have a particularinterest in the release of the peptide galanin in the ventral tegmentalarea because it has been hypothesized to represent an important linkbetween the noradrenergic and dopaminergic systems (see Weiss, J. M.,Demetrikopoulos, M. K., West, C. H. K. and Bonsall, R. W., Hypothesislinking the noradrenergic and dopaminergic systems in depression.Depression 3: 225245, 1996).

A previous method for estimating release of peptides in brain waspush-pull cannulation in which samples of extracellular fluid werecollected with a double, concentric cannula. The rate of fluid inflowhad to be exactly balanced against the rate of fluid withdrawal. Howeverflow rates were between 10 and 100 times higher than those commonly usedin microdialysis, and the procedure often created lesions that made theinterpretation of results difficult.

To solve this problem, the present invention includes a pushpull/microdialysis hybrid that is used in conjunction with the turntableto sample extracellular levels of the peptides. Probes built for thispurpose resemble concentric microdialysis probes but lack membranes. Thekey is to regulate the inflow and outflow at about 0.5 μl/min in such away as to produce a zero pressure difference between brain andperfusate. The use of low pressure delivery and sampling systemsminimizes the possibility of creating brain lesions, but moresophisticated remote sensing of the pressure and control of withdrawalrate may often be necessary. The sample collector consists of a rotatingvalve that diverts fixed volumes of sample into a storage coil.

Simultaneous Electrophysiology and Microdialysis

One of the most exciting possibilities presented by the turntable deviceof the present invention is the simultaneous use of electrophysiology tomonitor neuronal activity and microdialysis to measure the release ofneurotransmitters. Many of the technical problems inherent inelectrophysiological studies on alert, freely moving animals (e.g.maintaining electrode position) have already been addressed by others(see Lemon R. Methods for Neuronal Recording in Conscious Animals. IBROHandbook Series: Methods in The Neurosciences, Vol. 4 (A. D. Smith ed.)John Wiley and Sons, New York, 1984).

Multiple floating-wire electrode assemblies (e.g. Kosobud, A. E. K.,Harris, G. C. and Chapin, J. K., "Behavioral associations of neuronalactivity in the ventral tegmental area of the rat," J Neurosci. 14(1994) 7117-7129) are particularly suited to the multichannel capabilityof the turntable, which eliminate the need for a commutator. Thus, thepresent invention preamplifies the signal using head-mounted op-amps andthen processes and digitizes the signal on the turntable. The digitaldata is then transmitted to a PC using IR or RF methods. One technicalproblem is the generation of electrical noise by the stepper motor(although this is located about four feet away from the unamplifiedsignal). Shielding or digital filtering helps, as might microsteppingthe motor, but if necessary, the signal can simply be chopped during theintermittent operation of the motor. Other embodiments employ cyclicvoltammetry, which is relatively simple to perform intracranialself-stimulation (ICSS) in experiments on reward systems.

Applications to Smaller or Larger Animals

The turntable of the present invention was initially designed for usewith laboratory rats, and has applications for animals in the 150 to 800g range. The inertia of the tether-sensor assembly becomes significantfor smaller animals such as hamsters and mice. However, there are somestudies in which smaller rodents might be preferred, for example, theuse of mice in neuroimmunology. For such purposes, miniaturization ofthe sensor is necessary. For larger animals such as primates andungulates, on the other hand, a larger, stronger tether could bedeveloped, but this may not be necessary. The novel designs for infusionpumps and sample collectors described above can be mounted in aself-contained back-pack as readily as on a turntable. This provides,for the first time, a relatively inexpensive and reliable means ofobtaining multisite samples of biological fluids from untethered animalsin social situations. Applications to studies with humans are alsopossible.

Multichannel Sample Collection & Drug Delivery System

The multichannel automatic sample collection and drug delivery system ofthe present invention includes each of the above described parts, i.e.,a turntable adjustable tether, a coil sample collection device, aconstant pressure perfusion device and a multichannel microdialysisprobe for each animal under observation.

What is claimed is:
 1. A sample collection device comprising:a) a coilof flexible tubing, one end for coupling to a sample site; b) a timer;and c) a pump coupled to the other one end of the coil, wherein atpreset intervals controlled by said timer said pump draws a sample fromthe sample site into the coil followed by a bubble of gas to keepsuccessive samples separate in the coil until the samples are removedfor analysis.
 2. The device according to claim 1, further comprising acooling device surrounding the coil.
 3. The device according to claim 2,wherein said cooling device comprises:a) a reverse thermocouple coolingsystem; b) a thermistor sensor; and c) a computer being coupled to thethermistor and controlling the reverse thermocouple cooling system. 4.The device according to claim 1, further comprising a gas reservoircoupled to one end of said coil.
 5. The device according to claim 4,wherein said gas includes at least one of the noble gases from the groupconsisting of helium, argon, neon, xenon, krypton, and radon.
 6. Thedevice according to claim 4, wherein said gas includes at least one ofthe group consisting of nitrogen, air, or oxygen.
 7. The deviceaccording to claim 1, further comprising a solid-state delay relaymaintaining successive samples separate by introducing gas bubbles atregular intervals.
 8. The device according to claim 1, furthercomprising a computer controlling a frequency of bubble introduction. 9.The device according to claim 8, further comprising a plurality ofsensors coupled to the computer for controlling a size of the bubble.10. The device of claim 1, further comprising:a) a membrane coupled tothe sampling site; and b) a microdialysis probe coupled to the membrane,said probe including:(i) a first channel for delivering perfusate to thesampling site, said first channel extending into the membrane; (ii) asecond channel for delivering a preservative to mix with the sampleimmediately after the fluid sample enters the probe; and (iii) a thirdchannel for coupling to the coil of flexible tubing and receiving thefluid sample, wherein the third channel has a relatively wide bore ascompared to the other channels.
 11. The device according to claim 10,further comprising a fourth channel for delivering a drug to the samplesite.
 12. The device according to claim 10, wherein the first channelfurther comprises a tube for delivering a drug with the perfusate to thesample site.
 13. The apparatus according to claim 10, wherein said borehas a diameter of approximately 250μ meters.
 14. The device according toclaim 10, wherein the preservative includes low-pH mobile phase used forHPLC for monoamines.
 15. The device according to claim 10, wherein thepreservative includes peptidase inhibitors, antibodies or proteins. 16.An automated fluid sampling system for obtaining samples from a livingorganism comprising:a) a coil of tubing; b) an indwelling cannulaattached to the living organism, and also attached to one end of thecoil, said indwelling cannula having a main cannula with a tip, a bodyand an concentric inner cannula; c) a first syringe attached to theother end of the coil; d) a second syringe filled with an anticoagulantand being coupled to the indwelling cannula via the concentric innercannula, wherein said second syringe delivers said anticoagulant via theconcentric inner cannula to a point about 1 mm from the tip of the maincannula; e) a third syringe being coupled to the indwelling cannula viathe body of the indwelling cannula, and delivering a preservativesolution into the body of the cannula; f) an external computertransmitting an activation signal; g) three stepper motors, one coupledto each of the syringes; and h) a microcomputer receiving the activationsignal from the external computer and controlling the three steppermotors according to the activation signal.
 17. The system according toclaim 16, wherein the system performs the following withdrawalsequence:a) the first syringe withdraws a sample at a rate faster thanthe delivery rate of the anticoagulant and preservative, whereby a totalvolume withdrawn=volume of preservative+volume of anticoagulant+internalvolume of cannula+sample volume; b) to backfill the cannula, the mainsyringe is withdrawn at a withdrawal rate=coagulant delivery rate,wherein total volume=internal volume of cannula; c) to perform ananticoagulant drip, there is no main syringe or preservative syringemotion, and the anticoagulant flow rate=about 1 μl/min by intermittentpulsing of the appropriate stepper motor.
 18. The device according toclaim 16, wherein samples are stored in the sample coil between bands ofanticoagulant.